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Quantum yield is a determinant for fluorescent protein (FP) applications and enhancing FP brightness through gene engineering is still a challenge. Green2, our de novo FP synthesized by microfluidic picoarray and cloning, has a significantly lower quantum yield than enhanced green FP, though they have high homology and share the same chromophore. To increase its quantum yield, we introduced an F145Y substitution into Green2 based on rational structural analysis. Y145 significantly increased the quantum yield (0.22 vs. 0.18) and improved the photostability (t 1/2 , 73.0 s vs. 46.0 s), but did not affect the excitation and emission spectra. Further structural analysis showed that the F145Y substitution resulted in a significant electrical field change in the immediate environment of the chromophore. The perturbation of electrostatic charge around the chromophore lead to energy barrier changes between the ground and excited states, which resulted in the enhancement of quantum yield and photostability. Our results illustrate a typical example of engineering an FP based solely on fluorescence efficiency optimization and provide novel insights into the rational evolution of FPs., (© 2020 The Protein Society.)